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All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.

The aim of our research was to determine phosphorylation status of leukemic blasts by flow cytometry. In this study, we stained cells with anti-AKT (pT308), anti-ERK1/2 (pT202/pY204), and anti-p38 MAPK (pT180/pY182) monoclonal antibodies. In addition, we stained cells with anti-CD45 and/or anti-CD34 to gate only blasts in our samples. Kolmogorov-Smirnov statistical test was used to compare expression of phosphorylated molecules to negative isotype control staining. D value greater than 0.2 (D > 0.2) was considered positive. We found Akt phosphorylation/ activation in 30/48 (62.5%), ERK1/2 in 25/48 (52.1%), and p38 MAPK in 18/48 (37.5%) AML samples (in concordance with results of other AML phosphorylation studies). Interestingly, of these 18 p38 MAPK positive cases, 16 (88.8%) were also Akt positive. Statistical analysis revealed significant correlation (P ¼ 0.0001 ; r ¼ 0.55) between Akt and p38 phosphorylation, and chi-square test showed significantly higher frequency of AKT activation in p38 phosphorylated samples (P ¼ 0.009). In conclusion, signal transduction pathways PI3K/Akt and MAPK are dysregulated in human AML. Activation of p38 accompanied by Akt activation could indicate possible interactions and crosstalk between two signaling cascades. However, this hypothesis remains to be elucidated by further experiments.

Hematopoietic stem cell (HSC) transplantation is a standard approach in the treatment of hematological malignant diseases. For the last 15 years the main source of cells for transplantation have been peripheral blood stem cells (PBSC). With the availability of hematopoietic growth factors and understanding the advantages of treatment with PBSC, the application of bone marrow (BM) was supplanted. The aim of this survey was to explore the success of PBSC collection, the factors which influence the success of PBSC collection, the composition and the quality of graft and their influence on hematopoietic recovery and outcome after transplantation in patients with acute myeloid leukemia (AML). PBSC were collected by the method of leukapheresis after applying a combination of chemotherapy and growth factors or only growth factors. The quality of graft was determined with the clonogenic progenitor cell assay and with the flow cytometry analysis. Of the total 134 patients with AML, who were submitted to HSC mobilization, the collection was successful in 78 (58.2%) patients. The collection was more successful after the first than after the second attempt of HSC mobilization (49% vs. 11%). The criteria for effective mobilization were the number of leukocytes > 3 x 10(9)/L and the concentration of CD34+ cells > 20 x 10(3)/mL in the peripheral blood on the first day of leukapheresis. The number of CD34+ cells infused had the strongest impact on hematopoietic recovery. We noted significantly faster hematological recovery of neutrophils and platelets, fewer number of transfused units of red blood cells and platelets, shorter duration of the tranfusion support, shorter treatment with intravenous antibiotic therapy and shorter hospitalization after PBSC compared to BM transplantation. These advantages could provide their standard application in the treatment of patients with AML.

nm23 was identified as a metastasis suppressor gene but is also appointed to a number of other biological functions. The goal of this study was to reveal the influence of ectopic expression of nm23-H1 and mn23-H2 on proliferation properties of head and neck tumor cells. The proliferation rate of transfected cells was evaluated using EGFP reporter system and flow cytometry. HEp-2 and CAL 33 cells transiently transfected with nm23 cDNA containing constructs exhibited enhanced proliferation. CAL 27 cells constitutively expressing GFP-Nm23-H2 protein, exhibited intense proliferation the first day after seeding, while the GFP-Nm23-H1 expressing clone started to proliferate after one-day lag period. The results on transiently transfected HEp-2 and CAL 33 cells generally confirmed previous findings connecting nm23 expression with altered proliferation of head and neck tumors. We speculate that the effects observed on stably transfected CAL 27 clones are due to their different attachment properties.

Background and purpose: GFP (green fluorescent protein) is widely used in a variety of fluorescent methods aimed at revealing the fate of proteins in the cell, intracellular transport, transfection efficiency and is also recommended for cell cycle analysis purposes. In our attempt to evaluate the role of nm23 genes in proliferation of head and neck tumor cells in culture we have decided to use EGFP reporter system and analyze the DNA content by flow cytometry. Materials and methods: To optimize the method we either transiently transfected the cells with pEGFPC1-nm23 constructs or cotransfected the cells with an nm23 carrying constructs and pEGFPC1 as a reporter system. We also established stable clones with pEGFPC1-nm23 constructs and analyzed them by flow cytometry, as well. Results and conclusions: We report our experience for the use of pEGFP reporter system and flow cytometry for determining cell-cycle distribution of transiently and stably transfected adherent tumor cells. We discuss, in brief, the protocol we used and the problems that appeared during our experiments – GFP bleaching, cell clumping and degradation and insufficient number of cells to be analyzed. In conclusion, we suggest useful tips how to avoid or minimize the technical problems of this method and improve the results and analysis.

Objective: To explore the balance between serum and synovial fluid levels of interleukin-10 (IL- 10) and IL-18 in children with juvenile idiopathic arthritis (JIA). Methods: Blood samples were obtained from 81 child with JIA and 18 control children. Synovial fluid samples were collected from 16 children with pauciarticular JIA. Concentrations of IL-10 and IL-18 were determined using commercial kits. Results: Patients with systemic JIA had higher serum levels of IL-18 than patients with other forms of JIA or control children, both during the active (median, range: 6065, 387 – 78750 pg/mL) and inactive (1523, 121 – 3270 pg/mL) phase of disease (ANOVA, P

Introduction: Regulatory T (TR) cells play a mayor role in the homeostasis of the immune system. Different populations of TR cells have been described, including so-called “ naturally occurring” CD4+CD25+ T cells and IL-10-producing TR1 cells. Animal studies have shown that removal or inactivation of CD4+CD25+ T cells can break natural self-tolerance ; leading to development of autoimmune diseases, while reintroduction of this subset of T cells prevented diseases. Although the recent reports confirmed the role of TR cells in various human pathological conditions but surprisingly little is know about the TR cells in juvenile idiopathic arthritis (JIA). Aim: To explore the level of TR cells in peripheral blood of patients with JIA in relation to disease severity. Patients and Methods: Thirty-four JIA patients (20 with oligoarticular, 9 with polyarticular, and 5 with systemic JIA) and 23 healthy children were enrolled. The diagnosis was established according to the ILAR criteria. Peripheral blood samples were obtained in active and inactive phase of the disease and analysed by means of flow cytometry. The relative and absolute numbers of CD4+CD25+ cells, as well the cells with presumed TR phenotype (CD4+CD25++ and CD4+CD25++CCR4+) were measured. The groups were compared using ANOVA, followed by Student-Newman- Keuls post hoc test. The α -level was set at 0.05. The study was approved by the Ethics Committee of the Zagreb University School of Medicine. Results: Children with the systemic type of JIA had significantly higher levels of CD4+CD25+ lymphocytes than children with other types of JIA or the healthy controls (P

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare type of T-cell lymphoma of CD3+CD8+ phenotype characterized by deep-seated skin nodules or plaques mimicking panniculitis, a result of neoplastic lymphocytes infiltrating the subcutaneous fatty tissue. We present a case of a 19-month year old boy with SPTCL diagnosed and successfully treated in our institution. Disease first presented with symptoms of high fever and painful erythematous nodule located below the umbilicus. Later on the infiltrates appeared on the face, legs, arms and the back of the body. As the most decisive in obtaining the diagnosis, skin biopsy showed atypical, small to medium-sized lymphatic cells infiltrating the deeper dermal layers as well as the subcutaneous adipous tissue surrounding the adipocytes. Immunohystochemical analysis showed neoplastic lymphocytes positive for CD2, CD3, CD5, CD7, CD8, Tia-1, granzyme B and perforine, and negative for CD20, CD34, TDT and CD56. No infiltration of blood vessels or epidermis was evident. Specific T-cell lymphomas protocol (EURO-LB 02) was then initiated which resulted with rapid regression of all general and local symptoms. The treatment was completed according to schedule and the child is now, 24 months after the initiation of the treatment, in complete remission.

Recent data indicate that the apoptotic process, mediated by the CD95/Fas cell surface receptor, may be impaired in activated lymphocytes of patients with relapsing-remitting multiple sclerosis (RRMS). The aim of this study was to analyse the expression of CD95/Fas on peripheral blood (PB) CD4+ and CD8+ T lymphocytes and apoptosis of PB lymphocytes in RRMS patients in the relapse of the disease, immediately before and after pulse corticosteroid therapy (PCT), and in healthy individuals. The study included 27 patients (23 F, 4 M) with RRMS diagnosed by McDonald's criteria and 31 healthy controls (25 F, 6 M). Mean age of the patients was 36.85 +/- 7.52 years, mean duration of the disease 4.14 +/- 4.89 years, and average EDSS scale 3.24 +/- 1.18. Mean age of the controls was 36.54 +/- 10.72 years. The proportions of CD95+ T lymphocyte subsets were analysed by the use of monoclonal antibodies (anti-CD4-PE/Cy5, anti-CD8-FITC and anti-CD95-PE, DAKO) and flow cytometry (FACSscan cytometer and CellQuest software, Becton Dickinson). Apoptosis of lymphocytes was analysed by the use of annexin-V-FITC and PI labeling (Becton Dickinson), as well as flow cytometry. For statistical analysis nonparametric Mann-Whitney test for independent samples and Wilcoxon test for dependent samples were used. Results were presented as median (M) and range. The p values < 0.05 were considered as statistically significant. The proportion of CD4+CD95+ T lymphocytes was significantly higher in patients before therapy (M= 47.90 ; range 32.50 - 61.50), as compared to patients after therapy (M= 40.60 ; range 30.60 - 51.80, p< 0.01), and to controls (M= 36 ; range 30.40 - 40.10, p< 0.01). The proportion of CD8+CD95+ T lymphocytes was significantly higher in patients before therapy (M= 26 ; range 16.80 - 39.10), as compared to patients after therapy (M= 19.30 ; range 11-30, p< 0.01), and to controls (M= 19 ; range 12.30 - 25.50, p< 0.01). Apoptosis of lymphocytes was significantly lower in patients before therapy (M= 1.30 ; range 1- 4.70), as compared to patients after therapy (M= 5.60 ; range 3.10 - 48.80, p< 0.01), and to controls (M= 6.10 ; range 3.10 - 11.40, p< 0.01). Our results indirectly show that the process of apoptosis mediated by the CD95/Fas receptor is impaired in activated lymphocytes of RRMS patients. They also show that in those patients PCT influences on CD95/Fas expression on PB T lymphocytes, as well as on the lymphocyte apoptosis.

Evaluation of the clinical utility of the novel heavy/light chain immunoassay for the assessment of multiple myeloma patients.The novel heavy/light chain allows accurate measurement of monoclonal immunoglobulins and can be more sensitive than standard techniques at detecting residual disease. Abnormal heavy/light chain ratios and immunoglobulin isotype pair suppression may have prognostic significance for multiple myeloma patients.

Lymphomas represent the third most common group of cancers in childhood and adolescence, mature B non Hodgkin's lymphoma (B-NHL) accounting for up to 60% of newly diagnosed patients. The diagnosis of specific entities of B-NHL is based on well-defined morphologic analysis, immunophenotyping, cytogenetics and molecular genetics, which determine the optimal treatment strategy. In adult population a major turning point in treatment of B-NHL has been achieved since rituximab, in combination with CHOP has improved the survival rate up to 19%. Rituximab is a chimeric monoclonal antibody that targets CD20, a transmembrane calcium channel expressed on normal and malignant B-cells that mediates cytotoxic, apoptotic and anti-proliferative effects. The effect of rituximab in pediatric population is still not well enough investigated. Based on morphology and immunophenotype of malignant cells, seven children with B-NHL in our institution were eligible for treatment with modified B-NHL-Berlin-Frankfurt-Münster (BFM)-95-based protocol with rituximab administered on day -5. The complete remission was achieved in all seven patients. Six patients are still in complete remission at least 12 months after having finished chemotherapy and one patient relapsed two months after the last cycle and subsequently died. Major adverse effects observed during treatment were prolonged B-cell depletion and myelosuppression. Rituximab in combination with B-NHL-BFM-95 protocol was otherwise well tolerated and proved to be effective in children and adolescents with B-NHL. The number of our patients is too small and the follow-up of a larger group of patients will help in defining the role of rituximab in the treatment of childhood B-NHL.